The overall goal of this proposal is to determine the etiology of the Cl- permeability defect in cystic fibrosis (CF) using patch-clamp and optical techniques on intact sweat ducts. We argue here that the Cl channel primarily implicated thus far in this defect is not the one directly involved in the reduced Cl- permeability in CF. Studies on cultured cells have led to conflicting, and frequently erroneous conclusions about the frequency and number of types of Cl ability to stimulate them with agonists. Evidence for differences in these channels between control and CF cells is also unreliable. Furthermore, the levels of Cl- conductance observed for cultured epithelial cells are many times lower than those which have been measured for the noncultured tissues. However, it is necessary for understanding the etiology of CF to try to assess what specific channels and/or transporters are affected in CF and the mechanism by which their function is altered at as close to a molecular level as possible. We show here that it is possible to make both patch-clamp and optical (SPQ fluorescence) measurements on cells in intact sweat ducts. Sweat ducts have been chosen as the ideal tissue to study for understanding the CF defect since they have enormous Cl- conductances that are reduced many-fold in CF, they are readily obtained from living control and CF subjects, and they are uncompromised by secondary effects of CF. The combination of patch-clamp and optical techniques provides the means both to analyze the functioning and regulation of the molecules responsible for conductive Cl- transport on an individual level as well as to rapidly screen a large number of cells for the effects on Cl- transport of changes in second-messenger levels, transport blockers, induced expression of proteins, genetic variations, etc. We have also proposed direct studies of CFTR in membrane vesicles and lipid bilayer membranes in order to assess whether this molecule is, by itself, a Cl- transporter. Preliminary results from measurements on intact sweat ducts suggest that a different channel than those proposed from studies on cultured cells is the major conductance pathway in sweat duct and is the most likely candidate for the reduced Cl- conductance observed in CF.